System and method for cell levitation and monitoring

ABSTRACT

Magnetic cell levitation and cell monitoring systems and methods are disclosed. A method for separating a heterogeneous population of cells is performed by placing a microcapillary channel containing the heterogeneous population of cells in a magnetically-responsive medium in the disclosed levitation system and separating the cells by balancing magnetic and corrected gravitational forces on the individual cells. A levitation system is also disclosed, having a microscope on which the microcapillary channel is placed and a set of two magnets between which the microcapillary channel is placed. Additionally, a method for monitoring cellular processes in real-time using the levitation system is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/121,646 filed Aug. 25, 2016, which represents the U.S. national stageof PCT International Application No. PCT/US2015/017705 filed Feb. 26,2015, which claims priority to U.S. Provisional Patent Application Ser.No. 61/944,707 filed Feb. 26, 2014 and further claims the benefit ofU.S. Provisional Patent Application No. 62/072,040 filed on Oct. 29,2014. The contents of these applications are incorporated by referenceherein in their entirety for all purposes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with Government support under contracts CBET1150733 awarded by the National Science Foundation and R01 A1093282, R15HL115556, R01EB015776-01A1, R21HL112114, R01 HL096795, and P01 HG000205awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND

This disclosure relates to the levitation of a heterogeneous populationof cells and, more specifically, to the separation of cells based ondifferences in magnetic susceptibilities between the cells and asuspending medium, and a balance between magnetic and correctedgravitational forces.

Magnetic levitation has been traditionally used for analyses ofdensities and magnetic susceptibilities of individual, macroscopicobjects and as a means effective in separating foods, determining thefat content in milk, cheese, and peanut butter, comparing a variety ofgrains on the basis of their intrinsic densities, guiding self-assemblyof objects, and characterizing forensic-related evidence. These earliermagnetic levitation-based experiments were performed using large setupsthat were not compatible with or geared towards microscopy.

A wide variety of cellular processes, both physiological andpathological, are accompanied by transient or permanent changes in acell's volumetric mass density or magnetic signature due to formation orquenching of intracellular paramagnetic reactive species, for examplereactive oxygen species (ROS) or reactive nitrogen species (RNS). Theseevents include cell-cycle stage, differentiation, cell-death(apoptosis/necrosis), malignancy, disease state, activation,phagocytosis, in vivo and ex vivo cell aging, viral infection, andspecific as well as non-specific responses to drugs.

SUMMARY OF THE INVENTION

There have been few attempts to measure with high precision the densityof single living cells. One such technology involves nanofabricated,suspended microchannel resonators that offers low throughput, and thenecessity to use a sophisticated pump mechanism to transfer cellsbetween fluids with different densities. Therefore, reliable toolsdesigned for high resolution, real-time monitoring and quantification ofmagnetic signatures and volumetric mass densities of cells will helpelucidate the intricate cellular mechanisms.

The present invention overcomes the aforementioned drawbacks byproviding a levitation system for cells that is compatible withmicroscopy devices and that separates cells based on a balance betweencorrected gravitational forces on the cell and magnetic forces inducedby magnets. The corrected gravitational force is the gravitational forceexerted on the cell, accounting for the comparative densities of thespecific cell in the specific medium (that is, the buoyancy of the cell)as is outlined in greater detail in the detailed description section,below.

In accordance with one aspect of the present invention, a method forseparating a heterogeneous population of cells is provided. The methodincludes the steps of loading a sample of the heterogeneous populationof cells into a microcapillary channel containing amagnetically-responsive medium (for example, a paramagnetic medium, adiamagnetic medium, or a solution containing radicals capable ofproducing a sufficient environmental difference for separation), placingthe microcapillary channel containing the sample of cells and themagnetically-responsive medium into a levitation system, and levitatingthe heterogeneous population of cells in the magnetically-responsivemedium. The levitation system used is made up of a set of two magnetsproducing a magnetic field, with a space between the two magnets that issized to receive the microcapillary channel. Additionally, thelevitation system includes a microscopy device that has a stage betweenthe set of two magnets on which the microcapillary channel is placed.The step of levitation of the cells occurs by balancing a magnetic forceapplied to each of the cells by the magnetic field of the magnets with acorrected gravitational force of the cells in themagnetically-responsive medium, which consequently separates theheterogeneous population of cells. The mechanism is contrary to thosepreviously practiced, which have been known to balance magnetic forcewith inertial and drag forces rather than balancing magnetic forces withcorrected gravitational forces.

In some specific forms, the heterogeneous population of cells may beselected from that of red blood cells, leukocytes, lymphocytes,phagocytes, platelets, cancer cells, and the like. It is also possiblefor the magnetically-responsive medium to be a paramagnetic medium andto comprise gadolinium or to be gadolinium based, where the medium canbe pure gadolinium, or allow for additional constituents.

In a further possibility, the individual cells within the heterogeneouspopulation of cells may be differentiated from others based on at leastone of their magnetic susceptibility and cell density created by a cellvariant. The cell variant may be caused by multiple differences betweenthe cells such as cell type, cell-cycle stage, malignancy, diseasestate, activation state, cellular age, infection state, cellulardifferentiation, apoptosis of the cell, and phagocytosis of the cell.

In some forms, the magnetic field gradient may be created usingelectrical magnets. These electrical magnets may create the gradientusing alternating currents. In some forms, the set of two magnets may betwo permanent magnets in an anti-Helmholtz configuration.

In some forms, the separation of the individual cells may occur to anequilibrium exhibiting a balance between gravitational forces andmagnetic forces on the individual cells.

Furthermore, it is possible for the separation of the population ofcells to be performed at the point of care, as the levitation systembeing used does not interfere with mobile devices that can be used forremote diagnostics.

In some forms of the method, the method may further include the step ofobserving the heterogeneous population of cells in real time using themicroscopy device and the microscopy device may provide various imagesof the heterogeneous population of cells over a duration of time.

Over this duration of time, further steps may be performed. For example,a physical environment of the heterogeneous population of cells may bealtered and a response of the heterogeneous population of cells as aresult of the physical environment may be observed. As another example,a treatment agent (such as for example, a drug or an antibiotic) may beintroduced into the heterogeneous population of cells and a response ofthe heterogeneous population of cells as a result of the introduction oftreatment agent may be observed. If a treatment agent is introduced,then the method can further include monitoring a continued response ofthe heterogeneous population of cells to establish the emergence ofresistance of the heterogeneous population of cells to the treatmentagent.

In some forms, individual cells in the heterogeneous population of cellsmay be individually monitored and tracked during the step ofobservation.

In some forms of the method, the step of observation may includemonitoring the heterogeneous population of cells during different phasesof the cell life cycle.

In some forms of the method, the heterogeneous population of cells maybe levitated in a patient sample and it is further contemplated that thepatient sample may blood. Of course, blood is only one example, and thepatient samples are not contemplated as being limited only to blood.

In some forms of the method, healthy cells may be separated fromunhealthy cells. For example, cancer cells may be separated from healthycells. As another example, red blood cells may be levitated to detectthe presence of type I diabetes.

In some forms of the method, during the levitation step, live cells inthe heterogeneous population of cells may be separated from dead cells.This separation of live cells from dead cells in the heterogeneouspopulation of cells may used, for example, to determine the efficacy ofa treatment agent or to determine the effect of a change in the physicalenvironment on the cells.

In other forms of the method, during the step of separation, differentmicroorganisms may be separated from one another.

In some forms of the method, a characteristic of at least some of theheterogeneous population of cells may be determined by a measured heightof the cells in the microcapillary channel. In this way unhealthy cellsmay be detected without comparison to reference healthy cells.

In a further aspect of the method, the levitation system may include afirst mirror on a first open side of the microcapillary channel and asecond mirror on a second open side of the microcapillary channel inwhich the mirrors are oriented at oblique angles relative to the pathbetween the mirrors. While levitating the cells, the method may furtherinclude the step of reflecting light from a light source within themicroscope with the first mirror through the sample of cells and towardsthe second mirror to allow for real-time analysis of the cellpopulation. It is also possible for the microscopy device to be anupright fluorescence microscope leveled horizontally on its side, toallow for imaging of the cell population that does not requirereflection of light using the mirrors. Additionally, the microscopydevice might be a side-viewing microscope, a cell phone camera, alensless charged-coupled device (CCD) or complementary metal-oxidesemiconductor (CMOS) system, or an inverted microscope

In accordance with another aspect of the invention, a method forreal-time interrogation of cells is provided. The method includesloading a sample of the heterogeneous population of cells into amicrocapillary channel containing a magnetically-responsive medium,placing the microcapillary channel containing the sample of cells andthe magnetically-responsive medium into a levitation system, levitatingthe heterogeneous population of cells in the magnetically-responsivemedium, and altering magnetic properties of the magnetically-responsivemedium. Again, the levitation system used is made up of a set of twomagnets producing a magnetic field, with a space between the two magnetsthat is sized to receive the microcapillary channel. Additionally, thesystem includes a microscopy device that has a stage between the set oftwo magnets on which the microcapillary channel is placed. Levitation ofthe cells occurs by balancing a magnetic force applied to each of thecells by the magnetic field of the magnets with a correctedgravitational force of the cells in the magnetically-responsive medium,which consequently separates the heterogeneous population of cells.

As described previously, it is possible that the heterogeneouspopulation of cells may be selected from that of red blood cells,leukocytes, lymphocytes, phagocytes, platelets, cancer cells, and thelike. It is also possible for the magnetically-responsive medium to be aparamagnetic medium and to comprise gadolinium or to be gadoliniumbased, where the medium can be pure gadolinium, or allow for additionalconstituents. In a further possibility, locally altering magneticproperties of the magnetically-responsive medium may be accomplished byexposing the magnetically-responsive medium to a low intensity laserbeam.

In some forms, the individual cells within the heterogeneous populationof cells may be differentiated from others based on at least one oftheir magnetic susceptibility and cell density created by a cellvariant. The variant can be caused by multiple differences between thecells such as cell type, cell-cycle stage, malignancy, disease state,activation state, cellular age, infection state, cellulardifferentiation, apoptosis of the cell, and phagocytosis of the cell.

Furthermore, it is possible that separation of the individual cellsoccurs to an equilibrium exhibiting a balance between gravitationalforces and magnetic forces on the individual cells.

Furthermore, the separation of the population of cells may be performedat a point of care, as the levitation system being used does notinterfere with mobile devices that can be used for remote diagnostics.

In some forms, the levitation system may further include a first mirroron a first open side of the microcapillary channel and a second mirroron a second open side of the microcapillary channel in which the mirrorsare oriented at oblique angles relative to the path between the mirrors.While levitating the cells, the method may further include the step ofreflecting light from a light source within the microscope with thefirst mirror though the sample of cells and towards the second mirror toallow for real-time analysis of the cell population. In some forms, themicroscopy device may be an upright fluorescence microscope leveledhorizontally on its side, to allow for imaging of the cell population inway that that does not require reflection of light using the mirrors. Inother forms, the microscopy device might be a side-viewing microscope, acell phone camera, a lensless charged-coupled device (CCD) orcomplementary metal-oxide semiconductor (CMOS) system, or an invertedmicroscope.

Again, the magnets might take a number of different forms. For example,the magnets could be a pair of permanent magnets in an anti-Helmholtzconfiguration. As another example, the magnets may be electricalmagnets. By applying an alternating current to the electrical magnets,the magnetic field gradient may be created.

In some forms of the method, the method may further include the step ofobserving the heterogeneous population of cells in real time using themicroscopy device and the microscopy device may provide various imagesof the heterogeneous population of cells over a duration of time.

Over this duration of time, further steps may be performed. For example,a physical environment of the heterogeneous population of cells may bealtered and a response of the heterogeneous population of cells as aresult of the physical environment may be observed. As another example,a treatment agent (such as for example, a drug or an antibiotic) may beintroduced into the heterogeneous population of cells and a response ofthe heterogeneous population of cells as a result of the introduction oftreatment agent may be observed. If a treatment agent is introduced,then the method can further include monitoring a continued response ofthe heterogeneous population of cells to establish the emergence ofresistance of the heterogeneous population of cells to the treatmentagent.

In some forms, individual cells in the heterogeneous population of cellsmay be individually monitored and tracked during the step ofobservation.

In some forms of the method, the step of observation may includemonitoring the heterogeneous population of cells during different phasesof the cell life cycle.

In some forms of the method, the heterogeneous population of cells maybe levitated in a patient sample and it is further contemplated that thepatient sample may blood. Of course, blood is only one example, and thepatient samples are not contemplated as being limited only to blood.

In some forms of the method, healthy cells may be separated fromunhealthy cells. For example, cancer cells may be separated from healthycells. As another example, red blood cells may be levitated to detectthe presence of type I diabetes.

In some forms of the method, during the levitation step, live cells inthe heterogeneous population of cells may be separated from dead cells.This separation of live cells from dead cells in the heterogeneouspopulation of cells may used, for example, to determine the efficacy ofa treatment agent or to determine the effect of a change in the physicalenvironment on the cells.

In other forms of the method, during the step of separation, differentmicroorganisms may be separated from one another.

In some forms of the method, a characteristic of at least some of theheterogeneous population of cells may be determined by a measured heightof the cells in the microcapillary channel. In this way unhealthy cellsmay be detected without comparison to reference healthy cells.

In accordance with yet another aspect of the invention, a levitationsystem for separating a heterogeneous population of cells is taught. Thesystem includes a set of two magnets producing a magnetic field, with aspace between the two magnets which is sized to receive a microcapillarychannel adapted to receive the heterogeneous population of cells, and amicroscopy device with a stage between the set of two magnets on whichthe microcapillary channel is placed.

Furthermore, the system may include a first mirror on a first open sideof the microcapillary channel and a second mirror on a second open sideof the microcapillary channel in which the mirrors are oriented atoblique angles relative to the path between the mirrors. It iscontemplated that, in some forms, the microscopy device may be anupright fluorescence microscope leveled horizontally on its side. Inother forms, the microscopy device may be for, example, a side-viewingmicroscope, a cell phone camera, a lensless charged-coupled device (CCD)or complementary metal-oxide semiconductor (CMOS) system, or an invertedmicroscope and so forth.

Again, the magnets in the system might take a number of different formsor configurations. For example, the magnets could be a pair of permanentmagnets in an anti-Helmholtz configuration. As another example, themagnets may be electrical magnets. By applying an alternating current tothe electrical magnets, the magnetic field gradient may be created.

These and still other advantages of the invention will be apparent fromthe detailed description and drawings. What follows is merely adescription of a preferred embodiment of the present invention. Toassess the full scope of the invention, the claims should be looked toas the preferred embodiment is not intended to be the only embodimentwithin the scope of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 details magnetic levitation-mediated screening and modulation ofcells. FIG. 1a is a schematic of magnetic cellular levitation andmanipulation in a magnetic field. FIG. 1b is a schematic of the forceson a cell due to differences in magnetic susceptibility of the cell andthe medium. FIG. 1c is a plurality of views of the magnetic levitationsystem. FIG. 1d is a front and side contour plot of the magnetic fieldgradient induced by anti-Helmholtz configured magnets. FIG. 1e is a cellculture image of RBCS in 40 mM Gd⁺ solution.

FIG. 2 details the characterization of cell separation using magneticlevitation. FIG. 2a is a density histogram of monocytes, lymphocytes,basophils, PMNs, eosinophils, and RBCs. FIG. 2b is a cell culture imageof fluorescently-labeled RBCs, PMNs, and lymphocytes that have undergonemagnetically-driven, density based separation in 30 mM Gd⁺. FIG. 2c is acell culture image of RBCs levitated in 20, 35, 50, and 100 mM Gd⁺. FIG.2d is a cell culture time-lapse image set of the separation of old andyoung RBCs. FIG. 2e is a fluorescently labeled cell culture image of oldand young RBCs at their equilibrium levitation height. FIG. 2f is agraphical representation of the analytical equilibrium time as afunction of equilibrium height of old and young RBCs. FIG. 2g is a cellculture time-lapse image set of the levitation of sedimented RBCs in themagnetic levitation device. FIG. 2h is a graphical representation of thetime-dependent location of RBCs levitating from the bottom surface ofthe microcapillary to their equilibrium point.

FIG. 3 details the static levitation of functionally-altered bloodcells. FIG. 3a is a fluorescently labeled cell culture image detailingthe changes in PMN density associated with PMA activation. FIG. 3b is alow magnification image of levitating activated and resting PMN, with aninset showing the shape and optical density differences. FIG. 3c is apictorial representation of the roundness differences between restingand activated PMN. FIG. 3d is fluorescently-activated sell seedingresults for resting and activated PMN. FIG. 3e is a cell culture imagedetailing the differences in confinement height between resting,activated, and GSH-treated PMNs. FIG. 3f is a cell culture image ofmagnetically-driven density separation of blood cells. The left panelshows PMNs, lymphocytes, and platelets. The right panel shows restingPMNs, an activated PMN (arrow), and two eosinophils (arrowheads). FIG.3g is a cell culture image 2 hours after levitation detailing thehomo-typical aggregation of PMNs. FIG. 3h is a cell culture imagedetailing the phagocytosis of salmonella by human PMNs.

FIG. 4 details the functional interrogation of magnetically-levitatedblood cells. FIG. 4a shows manipulation of a cell or discrete group ofcells via localized laser irradiation. FIG. 4b is a cell culture imageset showing an increase in confinement height with UV stimulation, andparmagnetic-mediated cell clustering (dashed circle.) FIG. 4c is a cellculture image set showing the changes in magnetic properties of RBCs dueto increased intracellular ATP, causing cell clustering and decreasedlevitation.

FIG. 5 details the versatility of magnetic levitation for clinical pointof care diagnosis. FIG. 5a shows the brightfield image of the levitationof breast cancer cells (TC), PMNs, and lymphocytes from diluted bloodspiked with TC. FIG. 5b is the fluorescent imaging of the levitatedcells, TC being the larger cells in the top row. FIG. 5c is a merge ofFIGS. 5a and 5b . FIG. 5d is a cell culture image of the levitation ofhealthy RBCs in the presence of 10 mM Na metabisulfate. FIG. 5e is acell culture image of the levitation of sickle cell RBCs in the presenceof 10 mM Na metabisulfate.

FIG. 6 is a schematic of one embodiment of the levitation system. FIG.6a is an annotated photograph of the levitation system. FIG. 6billustrates the dimensions of some of the supporting elements.

FIG. 7 is a graphical representation of the equilibrium height as afunction of density difference between the suspending cell and theparamagnetic medium.

FIG. 8 is a micrograph of blood cells spiked with non-small cell lungcancer (NSCLC) cells (HCC-827) and the corresponding observed celldistribution profile. For this experiment, RBCs were previously lysedand cells were sorted using 30-mM Gadavist (Gd⁺) solution.

FIG. 9 is a micrograph of a non-small cell lung cancer cell (NSCLC)detected from a patient blood sample.

FIG. 10 illustrates the separation efficiency of breast cancer cells.FIG. 10a is a micrograph of fluorescently-labeled breast cancer cells(MDA) spiked in a blood. FIG. 10b shows the separation efficiency ofbreast cancer cells spiked in a blood sample with differentconcentrations in which the data points represent the mean of threereplicates with ±error bars standard deviation.

FIG. 11 illustrates the observed profile of peripheral blood mononuclearcells (PBMCs) of healthy and leukemia patients.

FIG. 12 illustrates the real-time density changes of single cells afterapplication to hydrochloric acid (HCl). FIG. 12a shows micrographs ofcontrol (untreated) and HCl-treated MDA breast cancer cells in which thecontrol cells maintain their levitation height (i.e., density), but theHCl-applied cells sink to the bottom of the channel (i.e., z=−500 μm).FIG. 12b details the real-time observation of a HCl-applied single cellin which a viability assay was also conducted using Calcein (greenfluorescent) for live cell and Propidium Iodine (red fluorescent) fordead cell. Fluorescent images and bright field images were overlappedeach other to compose the micrographs at different time point. While thecell is sinking through the channel bottom and it is gaining density,the fluorescent profile on the cell is changing from green to redindicating a dying cell. FIG. 12c shows real-time density measurement ofacid-treated single cells in which it is illustrated that, even if theacid is applied to the cells at the same time, each of the cells behavedifferently due to cellular heterogeneity.

FIG. 13 illustrates comparative height distribution profiles obtainedusing the magnetic levitation-mediated platform of untreated andampicillin-treated bacteria (i.e., E. coli) cells, in terms of magneticlevitation heights. Live and dead bacterial cells have distinct magneticprofiles that can be detected rapidly in real-time.

FIG. 14 illustrates magnetic levitation and characteristics of yeastcells. FIG. 14a graphs the viability of yeast cells after different drugtreatment for 24 hours. FIG. 14b illustrates how the levitation heights,magnetic properties and intrinsic magnetic signatures of yeast cells arealtered after drug treatment with 100 μM cantharidin and 100 μMfluconazole.

FIG. 15 provides data relating to the observed drug responses andobserved changes in magnetic profile of cells. FIG. 15a shows opticaldensity (OD) profiles, FIG. 15b shows distribution inside the channel,FIG. 15c shows calculated single-cell densities, and FIG. 15d providesvarious micrographs of cells treated with different concentration ofdrug (Fluconazole), the treatment concentration being listed above eachmicrograph. It is observed that cellular magnetic profiles and densitieschange after treatment with different drug concentrations and thesechanges can be monitored with the magnetic levitation system at thesingle-cell level.

FIG. 16 illustrates that budding yeast cells have different densitiesduring different phases of cell cycle and have distinct magneticprofiles. Mass, density, and volume of cells (i.e., Saccharomycescerevisiae) through the cell cycle can be measured by monitoring theirlevitation heights.

FIG. 17 provides magnetic profiles collected illustrating cellularsenescence and aging. The younger yeast cells are isolated bycentrifuging at 1400 rpm, while the older cells are isolated bycentrifuging at 900 rpm and 1400 rpm, respectively. Once they areisolated at the respected centrifugation speeds, the disclosed cellularlevitation device has shown that young and old cell populations havedifferent cellular magnetic profiles and levitate at different heights.

FIG. 18 details how the cellular levitation device can be used toidentify microorganisms. Yeast and bacteria cells are shown in FIG. 18to have different characteristic magnetic profiles; accordingly,individual microorganisms can be identified and separated from a mixedculture at low concentrations according to their densities using themagnetic levitation-based platform.

FIG. 19 provides micrographs and corresponding magnetic profiles ofhealthy and hepatitis C virus (HCV) infected hepatocytes. Infected cellsare clearly distinguished from healthy cells by density and magneticprofiles.

FIG. 20 provides magnetic profiles of RBCs of non-diabetic and diabeticmice (type I diabetes).

FIG. 21 illustrates changes in magnetic levitation profiles duringantibiotic treatment. E. coli cells were treated two hours withdifferent classes of antibiotics; 1 mg/mL of ampicillin (beta-lactamantibiotic) as in FIG. 21a , ciprofloxacin (fluoroquinolone antibiotic)as in FIG. 21b , and gentamicin (aminoglycoside antibiotic) as in FIG.21 c.

FIG. 22 illustrates changes in magnetic levitation profiles ofmulti-drug resistant bacteria during antibiotic treatment. The effectsof different antibiotics including ampicillin, ciprofloxacin, andgentamicin, as in FIGS. 22a, 22b, and 22c , respectively, on multi-drugresistant E. coli was investigated using the magnetic levitation system.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides a technique for real-time interrogationand monitoring of biological functions of magnetically-suspended cells.To achieve this, heterogeneous populations of cells are levitated andconfined in a microcapillary channel, for example a tube, placed betweentwo magnets, for example a pair of permanent magnets in anti-Helmholtzconfiguration. This enables equilibration of cells at different heightsbased on the balance between magnetic and corrected gravitational forcesacting on cells.

Permanent magnets in the setup can make this system easier to bereplicated and used by biomedical labs who would have interest in itsbroad applications. Using the permanent magnet system, constant magneticfield lines are created and thus, a minimum magnetic field strengthlocation which is spatially constant and dictates the levitation heightsof cells. By using alternating current, the magnetic field can bechanged in direction and intensity, as well as minimum field strengthlocation. Alternating magnetic field in principle may add newcapabilities such as changing levitation height of cells over the time.

Using this approach, red blood cells, leukocytes, platelets andcirculating metastatic breast cancer cells, as well as red blood cellsof different ages are separated. In addition, cellular processes such asneutrophil activation, phagocytosis, and responses of healthy and sicklered blood cells to dehydration are monitored in real-time. Thistechnique provides a broadly applicable tool for high resolution,real-time cell biology research, as well as disease screening anddiagnostics for point-of-care settings.

The core principle of the present magnetic levitation-based methodrelies on the equilibrium of two opposing forces: the correctedgravitational force and the magnetic force. What is presented is apowerful magnetic levitation-based microfluidic platform, which allowsreal-time, label-free, high resolution monitoring of cell populations,and is fully compatible with upright or inverted microscopes. Thistechnology offers rapid separation of different cell populations basedon their magnetic signatures and densities without the use ofantibody-tagged magnetic beads, centrifugation or the use of aspecialized, continuous or discontinuous density gradient media. Thelevitation platform enables unique monitoring functional responses ofindividual cells to a variety of stimuli, over time, and on acell-by-cell basis. This approach allows us for the ex vivoinvestigation of the biological responses following specific, cell-celland cell-molecule interactions in quasi-physiological, blood stream-likesettings.

The underlying mechanisms for levitation of cells in a microcapillarycan be understood as follows. Under an applied magnetic field, B,created by the two magnets placed in anti-Helmholtz configuration (samepoles facing each other), magnetic force, F_(m), exerted on a cell isgiven in Equation 1. Corrected gravitational force, F_(g), acting on acell is given in Equation 2

$\begin{matrix}{F_{m} = {( \frac{\chi_{cell} - \chi_{m}}{\mu_{0}} ){V( {B \cdot \nabla} )}B}} & (1) \\{F_{g} = {( {\rho_{cell} - \rho_{m}} ){Vg}}} & (2)\end{matrix}$

Here, μ₀=4π×10⁻⁷ (N·A⁻²) is the magnetic permeability of free space,ρ_(m) (kg·m⁻³) is the density of the paramagnetic medium, χ_(m) is thenon-dimensional magnetic susceptibility of the paramagnetic medium,ρ_(cell) (kg·m⁻³) is the density of the cell, χ_(cell) is thenon-dimensional magnetic susceptibility of the suspended cell, V (m³) isthe volume of the cell, and g is the vector of gravity. The cell isassumed to have a homogeneous distribution of density and magneticsusceptibility throughout its volume.

The magnetic force, F_(m), depends on the position of cell (as themagnetic field spatially changes within the microcapillary) and isdirected towards the minimum of the magnetic field. The correctedgravitational force, F_(g), does not depend on the location of the cellinside the microcapillary. The Stoke's drag force, F_(d) is given byequation 5 for a spherical particle of radius, R, and volume, V=4πR³/3.

In a transient case, for example before cell reaches equilibrium pointwhere the magnetic force balances with the corrected gravity force,inertial forces, for example the term at the left in Equation 3, anddrag force, F_(d), which depends on the migration velocity of cell,equation 5, will be active as described in equation 3. At equilibrium,the drag and inertial forces vanish, and the magnetic and gravitationalforces acting on the cell will balance each other, as given in Equation4.

$\begin{matrix}{\mspace{79mu}{{m\; a} = {F_{m} + F_{g} + F_{d}}}} & (3) \\{\mspace{79mu}{{F_{g} + F_{m}} = {{{( {\rho_{cell} - \rho_{m}} )Vg} + {( \frac{\chi_{cell} - \chi_{m}}{\mu_{0}} ){V( {B \cdot \nabla} )}B}} = 0}}} & (4) \\{\mspace{79mu}{F_{d} = {6\;\pi\;\eta\; R\; v}}} & (5) \\{\mspace{79mu}{F_{g} = {{( {\rho_{cell} - \rho_{m}} )Vg} = \begin{pmatrix}0 \\0 \\{{- ( {\rho_{cell} - \rho_{m}} )}Vg}\end{pmatrix}}}} & (6) \\{F_{m} = {{( \frac{\chi_{cell} - \chi_{m}}{\mu_{0}} ){V( {B \cdot \nabla} )}B} = \begin{pmatrix}{( \frac{\chi_{cell} - \chi_{m}}{\mu_{0}} ){V( {{B_{x}\frac{\partial B_{x}}{\partial x}} + {B_{y}\frac{\partial B_{x}}{\partial y}} + {B_{z}\frac{\partial B_{x}}{\partial z}}} )}} \\{( \frac{\chi_{cell} - \chi_{m}}{\mu_{0}} ){V( {{B_{x}\frac{\partial B_{y}}{\partial x}} + {B_{y}\frac{\partial B_{y}}{\partial y}} + {B_{z}\frac{\partial B_{y}}{\partial z}}} )}} \\{( \frac{\chi_{cell} - \chi_{m}}{\mu_{0}} ){V( {{B_{x}\frac{\partial B_{z}}{\partial x}} + {B_{y}\frac{\partial B_{z}}{\partial y}} + {B_{z}\frac{\partial B_{z}}{\partial z}}} )}}\end{pmatrix}}} & (7)\end{matrix}$

Here, v is the velocity of the particle (m/s) and η is the dynamicviscosity of the suspending medium (kg/ms). In the z-axis, where thecorrected gravitational force is aligned, the balance of forces can bewritten as,

$\begin{matrix}{{{( {\rho_{cell} - \rho_{m}} )Vg} + {( \frac{\chi_{cell} - \chi_{m}}{\mu_{0}} ){V( {{B_{\chi}\frac{\partial B_{Z}}{\partial x}} + {B_{y}\frac{\partial B_{Z}}{\partial y}} + {B_{Z}\frac{\partial B_{Z}}{\partial z}}} )}}} = 0} & (8)\end{matrix}$

Here, it is assumed that the absolute value of the third term

$( {B_{z}\frac{\partial B_{z}}{\partial z}} )\;$

in Equation 8 is larger than the absolute value of the sum of the firstand second terms

$( {{B_{x}\frac{\partial B_{z}}{\partial x}} + {B_{y}\frac{\partial B_{z}}{\partial y}}} )\;,$

and a linear change of B_(z) with respect to z-axis, therefore

$\begin{matrix}{{B_{Z}\frac{\partial B_{Z}}{\partial z}} ⪢ ( {{B_{x}\frac{\partial B_{Z}}{\partial x}} + {B_{y}\frac{\partial B_{Z}}{\partial y}}} )} & (9) \\{{B \equiv \begin{pmatrix}B_{x} \\B_{y} \\B_{z}\end{pmatrix}} = \begin{pmatrix}0 \\0 \\{{{- \frac{2B_{0}}{d}}z} + B_{0}}\end{pmatrix}} & (10)\end{matrix}$

Equation 8 can be solved after substituting B, given by Equation 10,into Equation 8 to find the equilibrium height, h, as seen in Equation11. Equilibrium height, h, is the vertical distance where the magneticforce and the corrected gravitational force cancel each other. Fromequation 11, ρ_(cell) can be extracted as well and written as a functionof h, Equation 12a, with the coefficients α and β, Equation 12b&c.

$\begin{matrix}{h = {\frac{( {\rho_{cell} - \rho_{m}} )g\mu_{0}d^{2}}{( {\chi_{cell} - \chi_{m}} )4\; B_{0}^{2}} + \frac{d}{2}}} & (11) \\{\rho_{cell} = {{\alpha h} + \beta}} & ( {12a} ) \\{\alpha = \frac{4( {\chi_{cell} - \chi_{m}} )B_{0}^{2}}{g\mu_{0}d^{2}}} & ( {12b} ) \\{\beta = {\rho_{m} - \frac{2( {\chi_{cell} - \chi_{m}} )B_{0}^{2}}{g\mu_{0}d}}} & ( {12c} )\end{matrix}$

Equilibrium height as a function of density difference between cell andthe suspending liquid is plotted in FIG. 7.

The time to equilibrium can also be calculated. Here, it is defined thatequilibrium time, t₀, is the time that elapses while a cell moves fromits initial location, z_(i) (for example, the bottom of themicrocapillary), to another position, z_(f) (for example the levitationheight), in the microcapillary. To find t₀, here it can be assumed inEquation 3 that the cell has zero acceleration (a=0) and is moving withits terminal velocity, as described by Equation 13.

0=F _(m) +F _(y) +F _(d)  (13)

The z component of equation 13 was found by substituting Equations 5, 8,and 10 into Equation 13, the substitution shown as Equation 14a. Afterintegrating Equation 14a, the time that elapses while a cell reachesequilibrium was found as described by Equation 15. While the cell getscloser to the equilibrium point, the driving magnetic force becomessmaller and thus, the velocity of the cell becomes smaller, which inturn decreases the drag force. In the mathematical model, the cell neverreaches equilibrium, therefore t₀=∞ when the Equation 15 is solved forz_(f)=h.

$\begin{matrix}{\frac{dz}{dt} = {{\xi z} + \zeta}} & ( {14a} ) \\{{\xi = {\frac{8}{9}\frac{R_{eq}^{2}B_{0}^{2}}{\mu_{0}d^{2}\eta}( {\chi_{cell} - \chi_{m}} )}}\;} & ( {14b} ) \\{\zeta = {{- \frac{2}{9}}\frac{R_{eq}^{2}}{\eta}( {{( {\rho_{cell} - \rho_{m}} )g} + {\frac{2B_{0}^{2}}{\mu_{0}d}( {\chi_{cell} - \chi_{m}} )}} )}} & ( {14c} ) \\{t_{0} = {\frac{1}{\xi}{\ln( \frac{{\xi z_{f}} + \zeta}{{\xi z_{i}} + \zeta} )}}} & (15)\end{matrix}$

Equilibration times of new and mature red blood cells are plotted as afunction of equilibrium height in FIG. 2h . In some experiments, themagnetic susceptibility of cell or the ambient paramagnetic fluid wasaltered by exposing it to UV and causing the formation of reactiveoxygen species (ROS).

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1: Magnetic Levitation Approach and Underlying Mechanisms

Negative differences between the magnetic susceptibilities of suspendingobjects, χ₀, (for example, a heterogeneous group of cells) andsuspending medium (χ_(medium)) create a magnetic force field causingobjects to be confined at different heights depending on the balancebetween corrected gravitational forces and magnetic forces as depictedin FIG. 1a . Negative difference between the magnetic susceptibilitiesof an object (χ₀) and suspending medium (χ_(medium)) causes the objectto move away from larger magnetic field strength site to lower magneticfield strength. Until an object, for example, a red blood cell (RBC)suspended in a paramagnetic medium reaches the equilibrium height, a setof forces, such as fluidic drag, inertial, gravitational, and magneticforces, continuously act on the object. As the object approachesequilibrium, its velocity, and thus drag and inertial forces becomeprogressively smaller, which can be seen in FIG. 1 b.

In this setup and with additional forward reference to the deviceillustrated in FIGS. 6a and 6b , a microcapillary tube that is 50 mmlong with a 1 mm×1 mm square cross-section is placed between twopermanent N52 grade neodymium magnets (NdFeB, 50 mm length, 2 mm width,and 5 mm height) in an anti-Helmholtz configuration (same poles facingeach other). These parts were assembled together using 1.5-mm-thickpolymethyl methacrylate (PMMA) pieces that were cut with a laser system(VLS 2.30 Versa Laser). Before each separate measurement, themicrocapillary channel was plasma treated for 2 minutes at 100 W, 0.5Torr (IoN 3 Tepla) and then placed between the magnets. Two mirrors areplaced at 45° (or other oblique angles) to image levitation heightsusing an inverted microscope (Zeiss Axio Observer Z1) under a 5×objective or 20× objective, to create a device compatible withconventional microscopy systems for high resolution spatiotemporalmonitoring of cells during levitation, which is detailed in FIG. 1c .Due to the particular placement of the two magnets, with the symmetricmagnetic field strength distribution with respect to each axis (FIG. 1d), the suspended cells levitate at a position that depends on both thelocation of minimum field strength and the ratios of the magneticsusceptibility and cellular density.

To test this setup, RBCs that have been isolated from a healthy donorare suspended in 40 mM gadolinium-based (Gd+) paramagnetic medium. Theparamagnetic solution used for all experiments presented here iscurrently employed for MRI investigations in humans, is non-toxic, andcompatible with human blood cells. Following 10 minutes of magneticconfinement, RBCs stably levitated at a height of approximately 300 μmfrom the bottom magnet, forming a small, wall-less, blood stream-likeassembly as seen in FIG. 1 e.

For higher resolution brightfield and fluorescence imaging of (20×, 40×and 60×), a mirror-free setup coupled to a fluorescence uprightmicroscope leveled on its side is used.

Example 2: Cell Separation by Magnetic Levitation

Mass density distribution of human blood cells varies between 1.055 and1.11 g/mL as illustrated FIG. 2a . Volumetric mass density, defined asmass per unit volume, is one of the most fundamental physical parametersthat characterize a cell. Several cellular events such asdifferentiation, cell death (apoptosis/necrosis), malignancy,phagocytosis, and cell-age cause permanent or transient changes in cellvolumetric mass density.

The cell-separation capability of the setup was assessed by magneticallyconfining isolated and fluorescently labeled RBCs, polymorphonuclearleukocytes (PMNs), and lymphocytes, as shown in FIG. 2 b.

To isolate PMNs, 40 mL of blood was obtained by venipuncture fromhealthy adult volunteers in accordance with the guidelines of theInstitutional Review Board (IRB) of Beth Israel Deaconess MedicalCenter, and after informed consent in accordance with the Declaration ofHelsinki. The blood was drawn into a 60 mL syringe containing 14 mL 6%Dextran T500 and 6 mL citrate solution. After 1 hour to allow forseparation, the buffy coat was obtained and layered on top of 15 ml ofFICOLL® (a neutral, highly branched, high-mass, hydrophilicpolysaccharide which dissolves readily in aqueous solutions, obtainedfrom GE Healthcare) and centrifuged at 350×g for 15 minutes. The pellet,consisting of PMNs, eosinophils and contaminating RBCs, was resuspendedin 25 ml of 0.2% NaCl for 45 seconds to lyse RBCs, followed by additionof an equal volume of 1.6% NaCl with continuous end-over-end mixing tobalance the salt solution. The suspension was centrifuged at 350×g for 5minutes, and the pelleted PMNs were washed and resuspended in 1 mLHBSS⁺⁺.

Results show that cells suspended in 30 mM Gd+ solution form distinctdensity and cell specific confinement bands populated by RBCs, PMNs andlymphocytes alone.

The effect of magnetic strength of the suspension solution on thefocusing height of RBCs was then investigated by progressivelyincreasing the molarity of Gd+ solution used for RBC suspension as canbe seen in FIG. 2c . It was found that an increase in the molarity ofGd+, and thus magnetic susceptibility of the suspension media, causes agradual increase in the focusing height of cells.

RBCs are formed in bone marrow by hematopoietic stem cells (HSCs) andcirculate for 100-120 days before they are recycled by tissuemacrophages. Circulating RBCs continuously releases microparticles thatprogressively decrease their surface-to-volume ratio and increase theirdensity.

To investigate if the sensitivity resolution of the setup was preciseenough to separate young (1.09 g/mL) from old (1.11 g/mL) RBCs based ontheir different volumetric mass densities, a mixture of fluorescentlylabeled young and old RBCs, which were isolated by PERCOLL® gradient(PERCOLL® being colloidal silica particles which have been coated withpolyvinylpyrrolidone), was levitated in 30 mM Gd+ solution.

To allow for fluorescent labeling, old and new RBCs were separated. RBCs(10% hematocrit) were collected either by venipuncture or fingerprickand washed 3 times in HBSS⁺⁺. RBCs were layered on 13 mL of a solutioncontaining 77% PERCOLL®, 10% 1.5 M NaCl, and 13% ddH₂O, followed bycentrifugation at 15000×g for 20 min, with the brake off. New RBCs atthe uppermost layer were collected, washed to remove the PERCOLL®solution, and resuspended in 1 mL HBSS⁺⁺. Similarly, old RBCs thatseparated to the bottom of the solution were collected, washed andresuspended in 1 mL HBSS⁺⁺.

RBCs, which were initially in random distribution in the microcapillary,started to focus at different levitation heights when exposed tomagnetic field (snapshots of the time lapse recording are shown in FIG.2d ). Fluorescently labeled young and old RBCs at their respectiveequilibrium levitation heights are shown in FIG. 2 e.

Using a time-lapse recording of the levitation process, the specificequilibrium time function of focusing height of old and young RBCs wasevaluated analytically as shown in FIG. 2f . Briefly, equilibriumheights were measured from the bottom magnet for young and old RBCs andwere found to be 0.156 mm and 0.092 mm, respectively. Densitydifferences between each cell and suspension liquid were calculatedusing FIG. 7, which was plotted using Equation 11. By substitutingdensity differences into Equations 14 and 15, equilibrium times wereplotted, which can be seen in FIG. 2 f.

The capability of the setup to levitate gravitationally-sedimented cellswas also tested. RBCs were loaded in the glass microcapillary tube, andthen placed on the bench for 15 minutes until all cells passively(gravitationally) sedimented along the bottom of the microcapillary. Themicrocapillary tube was then loaded in the magnetic levitation setup.Due to their relative diamagnetic properties compared to suspensionliquid, cells started to move away from the magnet and levitate towardtheir density-dependent equilibrium point, as shown in FIG. 2g .Finally, the location of cells during magnetic focusing as a function oftime using time-lapse microscopy was quantified, as seen in FIG. 2 h.

Example 3: Static Levitation of Functionally-Altered Blood Cells

PMNs are phagocytes, cells capable of sensing and responding tomicroorganism-specific danger signals followed by specific binding andinternalization of foreign microorganisms or particles. Phagocyticevents result in the formation of reactive oxygen species (ROS) andROS-mediated activation of hydrolytic enzymes. Generation of ROS andreactive nitrogen species (RNS) will cause changes in the magneticsignature of phagocytes, whereas the dynamic interplay between theendocytic and exocytic processes during phagocytosis would directlyimpact the volumetric mass density of activated PMNs.

Freshly isolated PMNs were activated by incubating them either withbuffer (resting PMN), GSH-ME (GSH-treated PMN), or 10 nM PMA (activatedPMN) for 5 minutes, washing twice, mixing, and resuspending them in 35mM Gd+ solution. Prior to treatments, cells were labeled either withCell Tracker Green (activated PMN) or Cell Mask Deep Red (GSH-treatedPMN).

The response of human PMNs during the activation phase of phagocytosiswas studied by incubating PMNs with phorbol 12-myristate 13-acetate(PMA, 10 nM) for 10 minutes. As a control, PMNs were left in buffer for10 minutes. Cells were then washed, fluorescently labeled, mixedtogether, and loaded into the magnetic levitation setup. Magneticfocusing revealed distinct differences between control and activatedPMNs, both in terms of size, shape, optical density, as well as magneticand mass density signatures, as shown in FIG. 3 a.

Activated PMNs generate intracellular paramagnetic ROS that activelyreduces the difference between the magnetic susceptibilities of thecells and suspending medium. As a consequence, activated PMNs would beexpected to “sink” compared to buffer-treated ones. However, the resultsshow that the decrease in density promoted by cell activation is morepronounced than the transient increase in magnetic properties and, as aresult, the cells levitated to higher elevations than the control.

The morphological differences between activated and normal PMNs wereevaluated by measuring the roundness of cells, defined as

${\frac{{perimeter}^{2}}{( {4 \cdot \pi \cdot {area}} )}.}\;$

The calculated roundness values, shown in FIG. 3c , indicate significantdifference between PMA-activated and buffer-treated PMNs. The samesamples were simultaneously examined by flow cytometry for changes inforward and side-scatter properties of PMNs associated with PMAactivation, shown in FIG. 3d . When compared to flow cytometry, magneticlevitation allows direct visualization of cells, as well as increasedshape and size-detection sensitivity and resolution, whilesimultaneously providing real-time density measurements on acell-by-cell basis.

To further understand the effect of intracellular ROS on the finalposition of levitating cells, cell permeable glutathione (GSH), an ROSscavenger was used. Results, depicted in FIG. 3e , show that GSH-treatedPMNs equilibrated closer to resting PMNs, whereas activated, low densityPMNs were focused as expected, above these two groups.

To test the density resolution of the setup, a mixture of PMNs,lymphocytes, and platelets was levitated. High magnification imaging ofthe resting PMNs revealed that, while most of the cells werenon-activated, a few, indicated by the arrow in FIG. 3f , show earlysigns of activation, through both shape changes and height positions,indicative of lower cell density. In addition, contaminating eosinophilswere positioned at the bottom of the PMN column, consistent with theirdensity being equal to or greater than that of the densest PMNs, as seenin FIG. 2a . Two hours after continuous levitation, as shown in FIG. 3g, PMNs underwent self-activation followed by integrin-mediatedhomo-typical aggregation.

Of note, some of the PMN clusters also displayed a lower positioncompared to non-activated PMNs, suggesting that intracellular,paramagnetic ROS species formed during activation also influenced theconfinement height of the cells. Next, the density changes during humanPMN phagocytosis were studied by incubating freshly isolated PMNs withfluorescently labeled Salmonella Montevideo. To allow for PMNphagocytosis, Cell Tracker Green-labeled PMNs (5×10⁵) were added tomicrofuge tubes containing 600 μL of HBSS/0.1% BSA. Serum-opsonizedAlexa-594-labeled S. Montevideo (1×10⁶), was added to the PMNs at a 10:1ratio, and the mixture was incubated for 10 min at 37° C. withend-over-end rotation at 8 rpm. PMNs were washed, mixed with Hoechst33342-labeled resting PMNs and resuspended in 35 mM Gd+ solution. TheSalmonella Montevideo (American Type Culture Collection) used was grownovernight in Bacto nutrient broth (Difco) and quantified (0.5OD₆₀₀=4.5×10⁸ cells/mL). Bacteria were gently pelleted, washed, andresuspended in HBSS.

A cell culture of this study can be seen in FIG. 3h . The results showthat phagocytic PMNs have significantly decreased density although therewas no clear relationship between the numbers of Salmonella ingested,originally shown as red in FIG. 3h (although now not readily apparentdue to the grey-scale conversion of the image), and the confinementheight of the PMNs.

Example 4: Functional Interrogation of Magnetically-Levitated BloodCells

The magnetic levitation setup permits acquisition of high resolutionimages at various points in time followed by investigation of uniqueresponses of individual cells in the population. This provides extensivemorphological and functional mapping capabilities over time on acell-by-cell basis for a given population.

The proposed platform allows for single cell manipulation by exposing aparticular area of confined cells to a low intensity laser beam, whichallows extensive spatiotemporal height adjustment of the targeted cellor cell group by transiently and locally altering the magneticproperties of the gadolinium solution, which is depicted in FIG. 4 a.

One levitation setup was fabricated using 1.5-mm-thick polymethylmethacrylate (PMMA) (McMaster Carr). Setup components were cut to thedimensions given in FIG. 6b (VERSALASER™; Universal Laser Systems Inc.).In this experimental setup, a microcapillary tube (VITROTUBES™ SquareCapillary Microcells, Borosilicate Glass 8100, Vitrocom, Mountain Glass,N.J.) with 1 mm×1 mm (outer edge, wall thickness 0.2 mm) squarecross-section is placed between two permanent neodymium magnets (NdFeB)in an anti-Helmholtz configuration (same poles facing each other). Twogold-coated mirrors are placed at each open side of the microcapillaryat 45 degrees to create a device compatible with conventional microscopysystems for high resolution spatiotemporal monitoring of cells duringlevitation. Microfluidic chips with easily accessible, inexpensivecomponents and magnets have been designed and fabricated, allowingwidespread use of this method by other researchers around the world.

In one experiment, following RBC confinement, a square area of 20×20 μmin the middle of stably levitated RBCs was illuminated with a 30 mW, 488nm laser beam at 0.34% intensity continuously for 1 minute using aVector Photomanipulation unit (3i). The targeting of the beam was kepton the same cell throughout the experiment.

In another experiment, a larger area (900×900 μm) of levitated RBCs,PMNs and lymphocytes was UV-irradiated. For the duration of irradiation,cells progressively increased their levitation heights due to increasedmagnetic properties of the suspension media. Immediately after UVstimulation was turned off, cells began to return to their originalpositions, although RBCs equilibrated at a lower height than original,indicating potentially that intracellular, UV-induced ROS increased theparamagnetic signature of RBCs. Cell cultures taken both whenUV-irradiation was on and off are depicted in FIG. 4 b.

Consistent with this possibility that UV-induced ROS increased theparamagnetic signature of RBCs, in areas with increased cell density,RBCs formed distinct aggregates, shown as the red circle in FIG. 4b ,likely due to weak paramagnetic attraction between ROS-containing RBCs.To test this hypothesis, a burst of extracellular ATP to dissociate 2-3DPG (2,3-diphosphoglycerate) from hemoglobin was used, a process thatalso transitions RBCs from diamagnetic to weak paramagnetic cells.Isolated RBCs were washed and resuspended in HBSS⁺⁺ containing 40 mM Gd+and 10 mM caged ATP (Life Technologies). RBCs were loaded into thecapillary, placed between the magnets, and allowed to focus. Next, aregion of 60×900 μm located about 70 μm above the levitating RBCs wasselected and illuminated by a 488 nm laser beam for 1 second at 100%intensity. The un-caged ATP, released into the suspension increased theextracellular concentration of ATP from 0 to nearly 10 mM. Cells wererecorded using Slidebook 5.5.

Following photolysis, caged-ATP became ATP, effectively increasing theconcentration of biologically-active, extracellular ATP from 0 to closeto 10 mM. The high concentration of extracellular ATP (10 mM) comparedto intracellular (about 1-1.3 mM) promoted an abrupt increase inintracellular ATP, followed by dissociation of 2,3 DPG from hemoglobin,and changes in the magnetic properties of RBCs that led toparamagnetic-mediated cell clustering, represented by the circles inFIG. 4c , and loss of levitation. As RBCs approached the bottom magnet,represented by the arrows in FIG. 4c , the interaction between theparamagnetic RBCs and the magnet increased progressively, eventuallyovercoming the weak paramagnetic interactions between cluster-formingRBCs, and leading to a gradual dispersal of RBC clusters.

Example 5: Versatility of the Magnetic-Levitation Based Approach forClinical POC Diagnosis

To demonstrate the wide applicability of this magnetic levitation-basedapproach over different cell types, circulating cancer cells, andsickled RBCs were used. Metastasis is a process responsible forspreading malignant cells from the primary site to another, non-adjacentsite. When malignant cells break away from a tumor, they migrate toother areas of the body through the bloodstream or the lymph system,becoming circulating tumor cells (CTC).

The breast cancer cell line MDA-MB-231 being used was purchased from theAmerican Type Culture Collection and cultured in DMEM supplemented with10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin andmaintained at 37° C. under 5% CO₂.

A heterogeneous group of cells by spiking normal blood with breastcancer cells (CTC) pre-stained with the cell permeable, DNA-specific dyeHoechst 33342 was prepared. The cell mixture was then magneticallyfocused for 15 minutes in a 20 mM Gd+ solution that allowed levitationonly of PMNs and lymphocytes, but not that of RBCs. CTCs were readilyidentified, shown as the cells with blue nuclei in FIG. 5a , in the toprow, from the multi-cell suspension being confined close to the centerof the microcapillary tube, tens to hundreds of micrometers away fromlymphocytes and PMNs respectively.

Additionally, it is shown that RBCs isolated from a healthy donor and apatient homozygous (SS) for sickle cell disease can be separated rapidlyand specifically based on their individual responses to 10 mM sodiummetabisulfite-induced dehydration, as can be seen in FIG. 5 c.

RBCs isolated from healthy and sickle cell disease patients were washedthree times and incubated with 10 μM sodium metabisulfite for 10 minutesat room temperature. Cells were levitated as described above and imageswere recorded after 10 minutes. To increase the contrast of the cellsagainst the background, images were filtered using an edge detectionalgorithm (Roberts). This treatment renders a subpopulation of sickleRBCs, likely younger RBCs, significantly denser than healthy RBCs.

Example 6: Label Free Detection of Circulating Tumor Cells (CTCs) andCirculating Tumor Microemboli (CTM)

With reference to FIGS. 8-11, label free detection of circulating tumorcells (CTCs) and circulating tumor microemboli (CTM) are illustrated.

With reference being made to FIG. 8, CTCs and CTM of non small cell lungcancer (NSCLC) were identified using the magnetic levitation system,where these cancer cells were levitated higher than the blood cellpopulation as illustrated in the left panel. In the right panel, acorresponding profile is illustrated which identifies the normalizedcell number at particular distances (i.e., levitational heights). Fromthe labeled peaks, it can be seen that the cancel cells and microemboliseparate upwardly from the white blood cells, which remain in at acomparatively lower height.

Turning now to FIG. 9, CTCs were also monitored also in NSCLC patientblood sample with the magnetic levitation system. The dotted whitecircle indicates a CTC in the patient blood sample.

Looking at FIG. 10, the separation efficiency of breast cancer cells isillustrated. It is demonstrated that the cancer cells spiked in bloodsamples can be separated in high efficiency from blood cells. In FIG.10a , a micrograph is provided in which fluorescently-labeled breastcancer cells (MDA) are spiked in blood and separated using thelevitation system. The inset panel of FIG. 10a shows three large dotswhich are the labeled MDAs. FIG. 10b illustrates the separationefficiency of breast cancer cells spiked in blood cells at differentconcentrations. This data establishes that the levitation system can beeasily applied for CTC and CTM quantification for cancer diagnostic andprognostic applications.

The device is also capable of identification of other cancer cells fromblood cells. For instance, as illustrated in FIG. 11, peripheral bloodmononuclear cells (PBMCs) of healthy and leukemia patients exhibitdifferent cellular profiles upon separation (i.e., the height of theleukemia cells is higher than that height of the PBMCs).

Example 7: Real-Time Monitoring of Response of Cells to theEnvironmental Factors in a Single Cell Level

As illustrated in FIG. 12, real-time monitoring of the response of cellsto the environmental factors can be performed at a single cell level. Inparticular, FIG. 12 illustrates the real-time density changes of singlecells after application to hydrochloric acid (HCl).

FIG. 12a shows micrographs of control (untreated) and HCl-treated MDAbreast cancer cells in which the control cells maintain their levitationheight (i.e., density), but in which the HCl-applied cells sink to thebottom of the channel (i.e., z=−500 μm) over 40 minutes of exposure to100 mM HCl.

FIG. 12b details the real-time observation of a HCl-applied single cellin which a viability assay was also conducted using Calcein (greenfluorescent) for live cell and Propidium Iodine (red fluorescent) fordead cell. Fluorescent images and bright field images were overlappedeach other to compose the micrographs at different time point. While thecell is sinking through the channel bottom and it is gaining density,the fluorescent profile on the cell is changing from green to redindicating a dying cell. This shows in real-time, over the span of 700seconds, the death of the cell and the correlation of this cell death toa density change and levitational height change.

FIG. 12c shows real-time density measurement of acid-treated singlecells in which it is illustrated that, even if the acid is applied tothe cells at the same time, each of the cells behave differently due tocellular heterogeneity.

By way of this example, it is illustrated how the effect ofenvironmental factors (e.g., pH, temperature, chemicals, and so forth)on cells can be monitored as cellular density changes. This can be usedto analyze cellular heterogeneity, which is helpful for understandingcancer, immune response, infectious diseases, drug resistance andevolution.

Example 8: Real Time Monitoring for Drug-Screening Applications

The levitation system also permits real-time assessment of cellularprofiles after drug treatment for drug-screening applications (i.e.,antibiotics, chemotherapy) as illustrated generally in FIGS. 13-15.Changes in the cellular profile during infections as well as during drugtreatment (i.e., antibiotics, anti-fungal drugs, anti-cancer agents) canbe observed rapidly using the magnetic levitation system.

As illustrated in FIG. 13 (and, in particular, the lower right panel ofFIG. 13), the magnetic levitation-mediated platform was used to detectsignificant differences between untreated and ampicillin-treatedbacteria (i.e., E. coli) cells, in terms of magnetic levitation heights.As the bacteria respond to the stimuli and antibiotics, their densityare altered. This directly reflects on their levitation heights.

Similar techniques can be used in real-time to monitor the emergence ofantibiotic-resistance in bacteria. Antibiotic resistance can be assessedby monitoring the live/dead bacteria as a function of their levitationheights that change in observed cellular density which are dynamicallyaltered post-exposure to antibiotics.

Looking at FIG. 14, the magnetic levitation and characteristics of yeastcells are illustrated that have either not been exposed or have beenexposed to various drug treatments for some duration of time. FIG. 14agraphs the viability of yeast cells after different drug treatments(i.e., no drug treatment/control, 100 μM cantharidin, or 100 μMfluconazole) for 24 hours. Different viabilities are observed andoptical densities are indicated for the various drug treatments. FIG.14b provides micrographs illustrating how, after 15 minutes of magneticlevitation, the levitation heights, magnetic properties and intrinsicmagnetic signatures of yeast cells are altered after drug treatment with100 μM cantharidin and 100 μM fluconazole for 24 hours.

Turning now to FIG. 15, this figure provides data relating to theobserved drug responses and observed changes in magnetic profile ofcells. FIG. 15a shows optical density (OD) profiles after various typesof drug exposure (i.e., control, 25 μM fluconazole, 50 μM fluconazole or100 μM fluconazole). FIG. 15b shows cellular distribution inside thechannel and FIG. 15c shows calculated single-cell densities. FIG. 15dprovides various micrographs of cells treated with differentconcentration of drug (fluconazole), the treatment concentration beinglisted above each micrograph. It is observed that cellular magneticprofiles and densities change after treatment with different drugconcentrations (both in height and spread, which are indicative ofhealth and variance in heath of cells, respectively). It is furthernoted that these changes can be monitored with the magnetic levitationsystem at the single-cell level.

With forward reference to FIG. 21, differences are illustrated in theobserved magnetic levitation profiles of E. coli cells after differenttypes of antibiotic treatment. The E. coli cells were treated for twohours with different classes of antibiotics including 1 mg/mL ofampicillin (beta-lactam antibiotic), ciprofloxacin (fluoroquinoloneantibiotic), and gentamicin (aminoglycoside antibiotic). The magneticlevitation profile of each of these antibiotics are compared to acontrol (i.e., untreated E. coli cells) in FIGS. 21a, 21b, and 21c ,respectively. This data illustrates that different antibiotic treatmentschange the levitation heights and cellular magnetic profiles in adifferent manner.

With additional forward reference to FIG. 22, differences areillustrated in the observed magnetic levitation profiles of multi-drugresistant E. coli cells after different types of antibiotic treatment.In these experiments, the effects of different antibiotics (ampicillin,ciprofloxacin, and gentamicin, respectively) on multi-drug resistant E.coli were investigated using the magnetic levitation system. Thisclinical isolate is resistant to ampicillin and ciprofloxacin, but issusceptible to gentamicin. Accordingly, there was no significant changein levitation heights and magnetic levitation profiles after two hourstreatment with 1 mg/mL ampicillin and ciprofloxacin as illustrated inFIGS. 22a and 22b , respectively. On the other hand, there was anoticeable change in the levitation heights and magnetic levitationprofiles after treatment with 1 mg/mL gentamicin for two hours as isillustrated in FIG. 22 c.

Thus, the magnetic levitation system has the potential to test theefficacy of antibacterial treatments and the magnetic levitation systemcan be used for antibacterial susceptibility testing applications.

Example 9: Real-Time Monitoring of Emergence of Drug-Resistance inCancer Cells

These same type of techniques can be used to, in real-time, monitor theemergence of drug-resistance in cancer cells: Drug resistance in cancercells can be assessed by monitoring the levitation heights that changein their cellular profiles observed during magnetic levitation that aredynamically altered post-exposure to anti-cancer agents. It iscontemplated that the efficacy of drug treatment can be alsoinvestigated using these real time methods.

Example 10: Real-Time Detection of Cellular Heterogeneity at theSingle-Cell Level

Notably, this technology enables the real-time detection of cellularheterogeneity at the single-cell level as illustrated in FIG. 12 andestablishes that different cells may respond differently as the resultof many cellular factors.

Likewise, this means that the heterogeneity of drug responses ofdifferent cells can be monitored in real-time at single-cell resolution.For example, real-time density measurement of acid-treated single cellswere conducted and variance in the response of the cells was observedgiven the single cell resolution. Even though the acid was applied tothe cells at the same time, each cells behaved differently due to thecellular heterogeneity as is acutely illustrated in FIG. 12 c.

Accordingly, using this real-time levitation system, it is contemplatedthat certain groups of cells may be first characterized and thentreated. This variance in cell behavior across a population of cells maybe intrinsic, but the ability to monitor the cells at a single cellresolution, in response to the variance of environmental factors or inresponse to variable treatment conditions, provides a complex andsophisticated way to study the way that a population cells respond whichcould be invaluable to a better understanding the underlying mechanisms,behaviors, and responses in these systems and provides a powerfulassaying tool.

Example 11: Detection and Separation of Live and Dead Cells

Further to the experimental results observed in FIGS. 12 and 13, liveand dead cells can be separated using this platform. This potentiallypermits for both detection and characterization of cell populations orpotentially the separation of the live and dead cell populations fromone another. Such detection and sorting might be incorporated intotesting protocols or to selectively obtain certain varieties of cellsamples.

Example 12: Profile of Cell Cycle and of Cellular Senescence and Aging

The system or platform also can be used as a tool to observe cell cyclesand may be used to characterize certain types of cells based on theirobserved behavior.

As illustrated in FIG. 16, budding yeast cells have different densitiesduring different phases of cell cycle. In addition, this platform hasdemonstrated that observed profiles of the cells (i.e., yeast cells)change during the cell cycle. For example, as illustrated in FIG. 16,yeast cells at M-phase had a greater levitation height than the cells atS-phase.

Further observed profiles are provided in FIG. 17 illustrating cellularsenescence and aging in yeast. As yeast cells age, many physical andbiological changes occur. For example, cell size increases, cell cycleslows down, cell shape is altered, cell nucleolus tends to be largerand/or more fragmented, and cells become sterile. The providedmicrographs of old yeasts and young yeasts (left and center panels,respectively) illustrate that younger and older yeast populations aredifferent profiles. These profiles can be further characterized as inthe rightmost panel in which the normalized cell number is provided atvarious the levitation heights (i.e., distances). Thus, two verydifferent profiles can be generated and compared as in the rightmostpanel to illustrate the health of a particular cell group and,furthermore, due to the real-time nature of the data collection, can beused to create sequenced profiles characterizing the aging of theobserved cell population.

Example 13: Microorganism and Pathogen Identification

As illustrated in FIG. 18, this platform and system can also be used toidentify microorganisms and pathogens. Individual microorganisms (i.e.,bacterial yeast, fungi, virus) can be identified and separated from amixed culture at low concentrations according to their magneticsignatures using the magnetic levitation-based platform. For example,yeast and bacteria cells have different characteristic magneticdistributions or profiles. As illustrated in FIG. 18, with the system inused, the middle of the channel includes primarily bacteria cells, whilethe bottom of the channel includes primarily yeast cells.

Example 14: Distinguishing Gram-Positive and Gram-Negative BacterialSpecies Using Magnetic Profiles

It is further contemplated that gram-positive and gram-negativebacterial species can be distinguished using magnetic profiles andcellular distributions. Gram-positive and gram-negative bacteria havedifferent surface properties. For example, gram-positive bacteria cellwall consists of a thick layer of peptidoglycan (20-80 nm) and teichoicacids. Gram-negative bacteria cell wall is much more complicated,composed of an outer membrane (7-8 nm) and a thin layer of peptidoglycan(1-3 nm). In addition, gram-negative bacteria have a higher lipid andlipoprotein content due to the presence of an outer membrane as well asthe lipopolysaccharides (LPS). Thus, gram-negative and gram-positivebacteria have different densities due to the different compositions ofcell walls and these differences can be detected and monitored inreal-time using magnetic levitation principles.

Example 15: Viral Infection Detection on Cells

With reference to FIG. 19, micrographs and corresponding magneticprofiles are provided of healthy and hepatitis C virus (HCV) infectedhepatocytes. Infected cells are clearly distinguished from healthy cellsby density and magnetic profiles. This data clearly shows that themagnetic separation profile of hepatocytes cells change significantlywhen they are infected with hepatitis C virus (HCV) as illustrated inthe lowermost panel.

Example 16: Early Diabetes Detection

Finally, other types of cellular changes can also be observed using theplatform. In FIG. 20, magnetic profiles of red blood cells ofnon-diabetic and diabetic mice (type I diabetes). Blood cells type Idiabetes mice show different cellular density profiles than the bloodcells from the healthy cells. Accordingly, this device offers a new wayfor early diagnosis of diabetes by monitoring changes in levitationheights.

In the examples above, the following antibodies and reagents wereutilized: Hoecsht 33342 (H1399, Molecular Probes, Eugene, Oreg.); Hank'sBalanced Salt Solution (14025-092), Cell Mask Deep Red plasma membranestain (C10046), Cell Tracker Green, CMFDA (C-7025), NPE-caged ATP(A-1048, Life Technologies, Grand Island, N.Y.); FICOLL® (17-5442-03),PERCOLL® (17-0891-01, GE Healthcare, Pittsburgh, Pa.); citrate 4% w/v(S5770), Dextran T500 (31392), Glutathione reduced ethyl ester (GSH-ME,G1404), Sodium metabisulfite (S9000), Sodium Chloride (S5886, Sigma, St.Louis, Mich.); Phorbol 12-myristate 13-acetate (PMA, 1201, Tocris,Bristol, United Kingdom); VITROTUBES™ Square Capillary Microcells,Borosilicate Glass (8100, Vitrocom, Mountain Glass, N.J.);Gadolinium-based (Gd+) paramagnetic medium PROHANCE® (BraccoDiagnostics, Princeton, N.J.); CRITOSEAL™ (a capillary tube sealantincluding vinyl plastic from Fisher Scientific, Pittsburgh, Pa.).

In the examples above, to load samples into a microcapillary tube,microcapillaries were simply dipped into the sample vials, and samplefilled into the capillary due to capillary forces. For each experiment,new microcapillary was used. Further, unless otherwise stated, cellswere resuspended in 200 μL of 40 mM Gadolinium solution and loaded in1.0×1.0 mm square microcapillary tubes (wall thickness 0.2 mm) bysuperficial tension action. Critoseal™ was inserted into either end ofthe microcapillary to prevent cells from drifting during analysis. Thecapillary was then loaded into a slot between the magnets and cells wereimaged using either QImaging Emc² EMCCD camera on an Olympus BX62microscope or a Qimaging EXi CCD camera on a Zeiss Axioscope microscope.For high-resolution images, a fluorescence microscope leveled on itsside was perfectly horizontally placed, and used a mirror-free magneticlevitation setup. The images were analyzed with Slidebook 5.5. (3i,Denver, Colo.), ImageProPlus 7 (Media Cybernetics, Rockville, Md.), andiVison 4.7 (Biovision, Exton Pa.).

By virtue of these examples, the versatility of the microfluidic,magnetic levitation platform that allows separation and activation ofcells, as well as monitoring and quantifying of various morphologicalattributes, specific cellular activities and agonist responses inreal-time has been demonstrated. The strategy presented here allowsexamination of temporal responses of cells to bioactive mediatorsintroduced by caged compounds (such as, for example, ATP). Theadvantages of the system include, but are not limited to (i) simpleworkflow, (ii) lack of sophisticated micro/nano-fabrication components,(iii) disposable designs with the possibility for autoclaveable reusablemodules, and (iv) multi-dimensional, real-time quasi-physiologicalinvestigation of dynamic cell:cell communications such asantigen-presenting cell:T cell and platelet:monocyte interactions.

The magnetic levitation device offers numerous biotechnologyapplications as well as a platform to study and monitor severalfundamental cellular behavior. It provides unique capabilities for cellbiology research where cell densities matter, and can reflect variousprocesses such as cell-cycle, phagocytosis, apoptosis, anddifferentiation. This system is also sensitive to magneticsusceptibilities of cells, and can thus be used for analysis ofhemoglobin degradation within the RBCs (for example, stored blood andsickle cells). The capability of monitoring several cellular activitiescan be also significant for drug discovery, toxicity testing, and singlecell testing. Real-time monitoring of levitating cells, followed byprotein and nucleic acid analyses, will potentially open avenues forresearch in unique signaling mechanisms present only during low gravityconditions.

This platform allows for measurements of cell densities (for example,RBCs, white cells) and separation of cells based on the balance betweencorrected gravitational force and counter-acting magnetic force.Simplicity, small size-scale and flexibility of the design make thesystem also compatible with mobile devices for telemedicine and use inresource poor settings for screening and diagnostics of malaria-infectedred blood cells and sickle cells. This strategy does not requireantibodies, advanced microscopy instrumentations or techniques forreliable diagnosis, nor the presence of microscopy specialists. Thisstrategy holds great promise for identification, isolation and in-depthomics data analyses of subpopulation of cells in a variety of normal andpathological conditions.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

1. A method for separating a heterogeneous population of cells, themethod comprising the steps of: placing a microcapillary channelcontaining a sample of the heterogeneous population of cells in amagnetically-responsive medium into a levitation system, wherein thesystem comprises a set of two magnets producing a magnetic field,wherein a space between the two magnets is sized to receive themicrocapillary channel; and a microscopy device having a stage betweenthe set of two magnets on which the microcapillary channel is placed;and levitating the heterogeneous population of cells in themagnetically-responsive medium by balancing a magnetic force applied toeach of the cells by the magnetic field of the magnets with a correctedgravitational force of the cells in the magnetically-responsive medium,thereby separating the heterogeneous population of cells; and obtainingan image of the heterogeneous population of cells using the microscopydevice and analyzing the image to characterize the heterogeneouspopulation of cells.
 2. The method of claim 1, wherein the heterogeneouspopulation of cells are differentiated from others in at least one oftheir magnetic susceptibility and cell density, and a cell variantcreates this difference, wherein the cell variant is selected from thegroup consisting of cell type, cell-cycle stage, malignancy, diseasestate, activation state, cellular age, infection state, cellulardifferentiation, apoptosis of the cell, and phagocytosis of the cell. 3.The method of claim 1, further comprising the step of separating theindividual cells to an equilibrium exhibiting a balance betweengravitational forces and magnetic forces on the individual cell.
 4. Themethod of claim 1, wherein the heterogeneous population of cells isselected from a group consisting of red blood cells, leukocytes,lymphocytes, phagocytes, platelets, and cancer cells.
 5. The method ofclaim 1, wherein the levitation system comprises a first mirror on afirst open side of the microcapillary channel and a second mirror on asecond open side of the microcapillary channel in which the mirrors areoriented at oblique angles relative to the path between the mirrors, andwherein the method further comprises the step of reflecting light from alight source within the microscope with the first mirror through thesample of cells and towards the second mirror.
 6. The method of claim 1,wherein the microscopy device is an upright fluorescence microscopeleveled horizontally on its side, a side-viewing microscope, a cellphone camera, a lensless charged-coupled device (CCD) or complementarymetal-oxide semiconductor (CMOS) system, or an inverted microscope. 7.The method of claim 1, wherein the magnetically-responsive medium is aparamagnetic medium and comprises gadolinium.
 8. The method of claim 1,wherein the method is performed at the point of care, and wherein themagnetic field does not interfere with mobile devices.
 9. The method ofclaim 1, wherein the magnetic field includes a magnetic field gradientcreated by electrical magnets using alternating currents.
 10. The methodof claim 1, wherein the set of two magnets are permanent magnets in ananti-Helmholtz configuration.
 11. The method of claim 1, furthercomprising the step of observing the heterogeneous population of cellsare in real time by the microscopy device, the microscopy deviceproviding various images of the heterogeneous population of cells over aduration of time.
 12. The method of claim 11, further comprising thesteps of altering a physical environment of the heterogeneous populationof cells and of observing a response of the heterogeneous population ofcells as a result of the physical environment.
 13. The method of claim11, further comprising the steps of introducing a treatment agent intothe heterogeneous population of cells and of observing a response of theheterogeneous population of cells as a result of the treatment agent.14. The method of claim 13, wherein observing a response of theheterogeneous population of cells as a result of the treatment agentincludes monitoring a continued response of the heterogeneous populationof cells to establish the emergence of resistance of the heterogeneouspopulation of cells to the treatment agent. 15-16. (canceled)
 17. Themethod of claim 11, wherein individual cells in the heterogeneouspopulation of cells are individually monitored and tracked during thestep of observation.
 18. The method of claim 11, wherein the step ofobservation includes monitoring the heterogeneous population of cellsduring different phases of the cell life cycle.
 19. The method of claim1, wherein the heterogeneous population of cells includes some cellsthat are infected with a virus.
 20. The method of claim 1, wherein theheterogeneous population of cells are levitated in a patient sample. 21.The method of claim 1, wherein analyzing the image to characterize theheterogeneous population of cells involves evaluating morphologicaldifferences between the heterogeneous population of cells.
 22. Themethod of claim 1, wherein analyzing the image to characterize theheterogeneous population of cells involves producing a cell distributionprofile over a range of levitational heights.